Quantinova sybr green pcr

Total RNA was isolated via RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the protocol described in [14 (link)]. First-strand cDNA was converted from 1 μg of the isolated total RNA using QuantiNova Reverse Transcription Kit (Qiagen, Germany). The expression profile was assessed via RT-qPCR analysis. Real-time PCR was performed with Bio-Rad CFX96 system (Bio-Rad, Hercules, CA, USA) with QuantiNova SYBR Green PCR (Qiagen, Germany) following the protocol as described in [15 (link)]. The primers (Supplementary Figure S5) were designed using Primer Blast from the National Center for Biotechnology Information (NCBI) and synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA). Three biological replicates were tested with three technical replicates performed on each sample. The data were analyzed using Bio-rad CFX Manager 3.1 software. The relative expression levels (2^−ΔΔCT) were calculated according to Livak’s method [16 (link)]. The reference genes used in this study were GAPDH and Actin.

Cells were treated with SeC for 6, 12, 24 h, and RNA was then extracted using an RNAzol RT kit (Life Technologies, Rockville, MD, USA). The cDNA was synthesized using the total RNA (2 μg) by iScript^TM cDNA Synthesis kit (Bio-Rad). Real-time quantitative PCR was performed using QuantiNova™ SYBR® Green PCR (Qiagen, Valencia, CA) on CFX96 PCR instrument (Bio‑Rad Laboratories, Inc., Hercules, CA, USA). The thermocycling conditions were as follows: preamplification 2 min at 95 °C, PCRs were followed by 40 cycles of 5 s at 95 °C and 10 s at 60 °C. The relative expression of each gene was calculated by the relative fold change between treated and control groups using the 2^−ΔΔCq method. The PCR primer sequences used in this study were as follows. Human GAPDH forwards, 5′-TGCACCACCAACTGCTTAGC-3′, and reverse, 5′-GGCATGGACTGTGGTCATGA-3′; human NRF2 (NFE2L2) forwards, 5′-TCTGACTCCGGCATTTCACT-3′, and reverse, 5′-GGCACTGTCTAGCTCTTCCA-3′; human KEAP1 forwards, 5′ CATCCACCCTAAGGTCATGGA-3′, and reverse, 5′-GACAGGTTGAAGAACTCCTCC-3′; human p62 (SQSTM1) forwards, 5′-TCCTGCAGACCAAGAACTATGACATCG-3′, and reverse, 5′- TCTACGCAAGCTTAACACAACTATGAGACA-3′; human NQO1 forwards, 5′-CGCAGACCTTGTGATATTCCAG-3′, and reverse, 5′-CGTTTCTTCCATCCTTCCAGG-3′; human xCT (SLC7A11) forwards, 5′-CCCAGATATGCATCGTCCTT-3′, and reverse, 5′-GCAACCATGAAGAGGCATGT-3′.

Total RNA was isolated with the RNeasy Mini Kit (Qiagen, Hilden, Germany). The RNA quality and concentration of the samples were measured with the NanoDrop™ 2000 (Thermo Fisher Scientific). 100 ng of total RNA was reverse-transcribed using the QuantiNova™ Reverse Transcription Kit (Qiagen) using SimpliAmp™ Thermal Cycler (Thermo Fisher Scientific). Real-time PCR was carried out using 700 nM primers. And QuantiNova SYBR Green PCR (Qiagen) on a StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific). Primers were selected with the tool Primer-BLAST. The following primer sequences were used: COL1A1 (FW: 5′-TGAGCCAGCAGATCGAGA-3′; REV: 5′-ACCAGTCTCCATGTTGCAGA-3′); GAPDH (FW: 5′-GCCGTCTAGAAAAACCTGCC-3′; REV: 5′-AAAGTGGTCGTTGAGGGCAA-3′); OC (FW: 5’-GCAGCGAGGTAGTGAAGAGAC-3′; REV: 5′-AGCAGAGCGACACCCTA-3′); OPN (FW: 5′-TGGAAAGCGAGGAGTTGAATGG-3′: REV: 5′-GCTCATTGCTCTCATCATTGGC-3′); RUNX2 (FW: 5′-AGCCTTACCAAACAACACAACAG-3′; REV: 5′-CCATATGTCCTCTCAGCTCAGC-3′). Data analysis was performed by the 2ΔΔCt method and using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal reference. Data are presented as mean fold change with respect to the control condition (bare PEEK).